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Mackie was born in New Orleans, Louisiana. Anthony graduated high school from the esteemed drama program at the North Carolina School of the Arts (NCSA), located in Winston-Salem, North Carolina. He also attended the New Orleans Center for Creative Arts (NOCCA). He is a graduate of the Juilliard School (Group 30), where he was a classmate of actors Tracie Thoms and Lee Pace.

Mackie has worked as an understudy for Jeffrey Wright and Don Cheadle in Suzan-Lori Parks play Topdog/Underdog. Mackie's first starring role in a feature film was in the 2004 Spike Lee film She Hate Me.

His brother Calvin Mackie, is an Associate Professor at Tulane University.

Anthony Mackie has been cast as Tupac Shakur alongside Jamal 'Gravy' Woolard as The Notorious B.I.G and Derek Luke as Sean Combs in the 2009 film Notorious. As of March 2008, Mackie was starring in three plays by playwright August Wilson at the John F. Kennedy Center for the Performing Arts in Washington, DC: "Ma Rainey's Black Bottom", "Fences", and "Jitney" -- all part of "August Wilson's 20th Century", a month-long presentation of 10 staged readings of Wilson's acclaimed "Century Cycle".

• Babylon(2009) (pre-production) .... Jeremiah
• Jesse Owens(2009) (pre-production) .... Jesse Owens
• Desert Flower(2008) (filming) .... Harold
• Notorious (2009) (post-production) .... Tupac Shakur
• The Great Observer (2008) (post-production) .... Buddy Bolden
• Bolden!(2008) (post-production) .... Buddy Bolden
• Eagle Eye(2008)
• The Hurt Locker(2008) .... Sergeant JT Sanborn
• Ascension Day(2007) .... Turner
• We Are Marshall(2006) .... Nate Ruffin
• Crossover (2006) .... Tech
• Heavens Fall (2006) .... William Lee
• Half Nelson(2006) .... Frank
• Freedomland(2006) .... Billy Williams
• The Man (2005) .... Booty
• Million Dollar Baby(2004) .... Shawrelle Berry
• Sucker Free City(2004) (TV) .... K-Luv
• Haven(2004) .... Hammer
• She Hate Me(2004) .... John Henry 'Jack' Armstrong
• The Manchurian Candidate (2004) .... Robert Baker
• Brother to Brother(2004) .... Perry
• "Law & Order: Criminal Intent.... Carl Hines (1 episode, 2003)
• Hollywood Homicide(2003) .... Killer 'Joker'
• Crossing(2003) .... Cass
• 8 Mile(2002) .... Papa Doc
• "As If .... Bar patron (1 episode)